The genes arcA encoding for the aerobic respiration control protein and iclR encoding the isocitrate lyase regulator are known to regulate the central carbon metabolism. ArcA is a global transcriptional regulator that regulates a wide variety of genes, while IclR is a local transcriptional regulator that regulates the glyoxylate pathway. ArcA is known to regulate the central carbon metabolism in response to oxygen deprivation and has no connection with IclR other than that it also regulates the glyoxylate pathway (24, 28, 29, 37, 38). In an earlier study the combined effect of ΔiclRΔarcA mutant strains on the central carbon metabolism has been observed. Increased fluxes were shown in the tricarboxylic acid (TCA) cycle and glyoxylate pathway and an interesting and surprising phenotype appeared when both genes where knocked out, namely the double mutant strain formed biomass with a yield that approached the maximal theoretical yield (4, 39).
Some compounds, such as GDP-fucose, are in high demand. The latter compound is indeed a precursor of fucosylated oligosaccharides such as fucosyllactose, fucosyllactoNbiose and lewis X oligosaccharides, or, of fucosylated proteins. These sugars are components of human mother milk in which they have anti-inflammatory and prebiotic effects and/or have applications in therapeutics as nutraceutical, anti-inflammatory agent or prebiotic, in addition, fucosylated proteins find applications in the pharmaceutics (5, 8, 27). However, an efficient method to produce the latter high-value compounds is still needed.
In addition GDP-mannose is also an intermediate of the pathway towards GDP-fucose. Interrupting the pathway prematurely leads to the accumulation of this compound, which is a precursor of mannosylated oligosacharides. These oligosaccharides find for example applications in the treatment of gram-negative bacterial infections, in addition, GDP-mannose is important for the humanization of protein glycosylations, which is essential for the production of certain therapeutic proteins (18, 30). Mannosylated oligosaccharides and mannosylated glycoconjugates are also used for drug targeting, for instance mannosylated antivirals can specifically target the liver and kidneys (7).
The present invention provides microorganisms which are genetically changed in such a manner that they can efficiently produce the latter compounds.
Moreover, the synthesis of pH sensitive molecules, such as—but not limited to —glucosamine, and organic acids, such as—but not limited to—pyruvic acid, succinic acid, adipic, sialic acid, sialylated oligosaccharides . . . are preferably produced at low pH, either to stabilize the product or for downstream processing reasons (4, 12, 40). Therefore, strains that can grow at low pH are beneficial for these production processes. E. coli is an organism that can adapt easily to various conditions, for instance it can easily adapt to the harsh pH conditions in the stomach, which is about pH 2 (14). Nonetheless, E. coli does not seem to grow at these conditions, but induces its acid resistance mechanisms in the stationary phase (40). During this phase the cell does not multiply anymore and therefore hampers productivity. Up to now, no solution was found to this problem. However, in the present invention, a genetically engineered microorganism is provided that can induce acid resistance mechanisms in the exponential growth phase, which is the phase that is mostly used for production of organic acids and pH instable products.